The present invention is relates to a method for purifying a .gamma.-glutamyl carboxylase (also referred to herein as .gamma.-carboxylase) from Conus snails and to a method for assaying the activity of this .gamma.-carboxylase.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by number and are listed numerically in the appended List of References.
The vitamin K-dependent .gamma.-carboxylation of glutamate residues was originally discovered as a novel post-translational modification in the blood coagulation cascade (1); some of the key clotting factors such as prothrombin must be .gamma.-carboxylated in order for proper blood clotting to occur. Somewhat later, this post-translational modification was also found in certain bone proteins (2). This modification was restricted to these rather specialized mammalian systems until a very unusual peptide, conantokin-G, was described from the venom of the predatory marine snail, Conus geographus (3). Conantokin-G is a 17-amino acid peptide that inhibits the N-methyl-D-aspartate receptor (4). Unlike most Conus peptides, which are multiply disulfide-bonded, conantokin-G has no disulfide cross-links but has five residues of .gamma.-carboxyglutamate residues; this remains the highest density of .gamma.-carboxyglutamate found in any functional gene product characterized to date.
Most of the biologically active components of the Conus venom are multiply disulfide bonded peptides (the conotoxins). These have been shown to be initially translated as prepropeptide precursors, which are then post-translationally processed to yield the mature disulfide-crosslinked conotoxin. Conantokin-G differs strikingly from most conotoxins not only in having .gamma.-carboxyglutamate residues, but also because it has no disulfide crosslinks. We report below an analysis of a cDNA clone encoding the conantokin-G precursor. Furthermore, we establish the probable function of one region of the precursor that is excised during the maturation of the functional conantokin-G peptide.
The presence of .gamma.-carboxyglutamate in a non-mammalian system was initially controversial because vitamin K-dependent carboxylation of glutamate residues had primarily been thought to be a highly specialized mammalian innovation. However, we have found that conantokin-G is only one member of a family of peptides; a variety of other conantokins have been found including conantokin-T and conantokin-R from two other fish-hunting cone snails (5, 6). All three peptides have a high content of .gamma.-carboxyglutamate (4-5 residues). .gamma.-Glutamyl carboxylase has been purified from mammalian sources (7, 8) and has been expressed both in mammalian and insect cell lines (9, 10). Recently it was shown that, as is the case in the mammalian system, the carboxylation reaction in Conus venom ducts absolutely requires vitamin K, and the net carboxylation increases greatly in the presence of high concentrations of ammonium sulfate. In these respects, the mammalian and the Conus .gamma.-carboxylation venom systems are very similar (11).
The propeptides of vitamin K-dependent blood coagulation proteins share extensive sequence similarity. This sequence is believed to interact with the carboxylase and constitutes the .gamma.-carboxylation recognition sequence (.gamma.-CRS). In this report, we analyze the conantokin-G precursor sequence for potential .gamma.-CRS sequences. The results described below identify a sequence present in the -1 to -20 region of the conantokin-G prepropeptide which, when covalently linked to the N-terminal of the substrate enzyme, stimulates carboxylation by the Conus enzyme.